Human monocytotropic ehrlichiosis (HME), caused by the obligately intracellular bacterium Ehrlichia chaffeensis, is an emerging, often life-threatening, tick-transmitted infectious disease in the United States. Studies from our laboratory and others have characterized the primary adaptive immune responses against ehrlichial infections. However, few investigations have addressed the generation and maintenance of memory immune responses. While CD4+ Th-1 mediated immunity is essential for protection against primary ehrlichiosis, a role for CD8+ T cells in immune-mediated pathology is implicated in fatal primary and secondary ehrlichial infections. Our previous study indicated that closely related ehrlichial species which differ in virulence also differ in the induction of protective memory immune responses. Our recent study suggested that persistent ehrlichial infection contributes to protective heterologous immunity by stimulating maintenance of effector memory T cell responses. Further the study indicated that persistent E. muris infection induces expansion of CD4+CD25+ T regulatory cells and enhanced production of TGF-1 in the spleen, which may facilitate ehrlichial persistence in the vertebrate host. The hypothesis underlying the proposed research plan for the K22 award is based on these findings. The objectives of the present research proposal are (i) to investigate the magnitude and quality (multifunctional state) of memory T cells generated and maintained during persistent bacterial infections and their role in protection, and (ii) to define the influence of CD4+CD25+ T regulatory cells and immunoregulatory cytokine TGF- on memory T cell responses, bacterial persistence and immune- mediated pathology induced during bacterial infections employing well characterized murine models of ehrlichiosis. Our hypotheses are (i) the quality of memory T cells generated (as determined by their ability to secrete multiple, specific cytokines) correlates with protection and (ii) CD4+CD25+ T regulatory cells and/or TGF- influence memory T cell responses and contribute to persistence of ehrlichial infections. Our long-term goal is to understand the components of memory immune responses that are critical for conferring protection against ehrlichiosis and to establish correlates of protection as a prerequisite to develop effective vaccines against ehrlichioses. The proposed study is expected to provide new insights into the functional state of T cells maintained during persistent bacterial infections. We will utilize murine models of persistent ehrlichiosis, fatal ehrlichiosis and heterologous protection to accomplish our objectives. We will develop multiparameter flow cytometry in order to characterize memory T cells for their ability to secrete multiple cytokines (IFN-?, TNF-a and IL-2). We will use antibody-mediated depletion of CD4+CD25+ T regulatory cells and antibody-mediated neutralization of TGF- to determine their influence on memory T cell responses and ehrlichial persistence. Human monocytotropic ehrlichiosis (HME) is highly prevalent in the south-central, southeastern and mid- Atlantic regions of the United States. To date, no vaccine is available against HME. Understanding the generation and maintenance of memory T cell responses is critical for rational design of a vaccine against ehrlichiosis as T cells contribute to both protection and pathogenesis.